Introduction:
Tuberculosis, the most common infectious disease with prevalence of 9.6
million globally. Most prevalent (23%) in India. Extrapulmonary
tuberculosis (EPTB) accounts for 20% of total burden of tuberculosis.
Rapid detection of Mycobacterium Tuberculosis (MTB) is essential for
effective disease management. CBNAAT (Cartridge Based NucleicAcid
Amplification Test) or GeneXpert MTB/RIF assay - novel diagnostic tool
to detect MTB and RIF resistance simultaneously. WHO recommends its
utility for non-respiratory samples also. Burden of EPTB and drug
resistance vary from place to place. Objective: Study was
conducted to gather information about burden of disease in our locality
and to assesutility of CBNAAT in detecting MTB and rifampicin
resistance in suspected EPTB cases. Methods: Retrospective analysis of
281 samples from suspected cases collected in falcon tubes and
processed using CBNAAT. Result:
Total of 281 extrapulmonary samples received, 67(23.8%) were positive
and 214(76.1%) were negative for MTB. Of 67 positives, RIF resistance
detected in 1(1.49%) case. Maximum number of MTB detected in the age
group 21-30 years (n=23, 34.3%). Among 165 males and 116 females, MTB
detected in 44(26.6%) and 23(19.8%) respectively. Out of 281 patients,
24(8.54%) were HIV positive. Of these 24, only 8(33.3%) found positive
for MTB. Among 257 non-HIV patients, MTB detected in 59(22.9%). Among
different samples received, maximum number were Pleural fluid
n=115(40.9%) and Maximum MTB positives found in FNAC (of lymphnodes)
samples [n=35(52.2%)]. Conclusion:
CBNAAT is a rapid test to detect MTB and rifampicin resistance
simultaneously in EPTB and it reduced the treatment abuse in suspected
cases.

Tuberculosis (TB) is the most common infectious disease and in
developing countries like India, it is major health problem. Due to
inadequate diagnostic assays it remains as a challenge to public
health. According to the WHO Tuberculosis report in 2015, there are 9.6
million people infected with TB. India has the maximum number of cases,
that is 23% of the total cases all over the world. Globally, an
estimated 3.3% of new TB cases and 20% of previously treated cases have
MDR-TB [1].

Extra Pulmonary TB (EPTB) can affect any organ in the body and it
accounts for 20% of total burden of tuberculosis globally. In majority
of cases it remains undetected for a longer time. A major hindrance to
the diagnosis of EPTB is the atypical presentation, often simulating
neoplasia and/or inflammatory disorders and clinical specimens of deep
organs are difficult to obtain. Adding to this, the Mycobacterium
Tuberculosis Bacilli (MTB) load is generally very low in
non-respiratory samples, therefore strongly affecting the sensitivity
of acid-fast microscopy[2]. Also quick and reliable laboratory
diagnostic methods for detecting tubercle bacilli in EPTB specimens are
not easily available. This adds to the increased rates of morbidity and
mortality in EPTB patients [3].

As the conventional laboratory methods are slow and cumbersome,
Foundation for Innovative New Diagnostics (FIND) introduced
cartridge-based nucleic acid amplification assay (GeneXpert
MTB/rifampicin [RIF]). It is a molecular test which is fully automated
and detects MTB directly from clinical samples within two hours as well
as RIF resistance which is the surrogate marker of MDR-TB conferring
mutations in 81 bp RIF resistance determining region (RRDR) of the rpoB
gene, which codes for a beta subunit of RNA polymerase of MTB, is the
genetic basis of RIF resistance [4].

In 2014, The GeneXpert MTB/RIF assay has been strongly recommended by
WHO for testing non-respiratory specimens from patients suspected of
having EPTB to diagnose MTB and multidrug-resistant TB over the
conventional tests (including microscopy, culture or histopathology).
The test is currently recommended as a ‘‘first
line’’ fast diagnostic test in high TB burden
countries like India[5,6].

Burden of EPTB and drug resistance vary from place to place, therefore
this study was conducted to assess the burden of EPTB and drug
resistance in our locality.

Materials
and Methods

Type and place of study: Study
was a retrospective observational record-based analysis conducted in
the RNTCP lab attached to the Department of Microbiology in Bowring and
lady Curzon hospital.

Sample collection: Total
of 281 clinically suspected EPTB cases were received in RNTCP lab from
various department of our hospital and other private hospitals. Few
details of the patients like Name, Address, Age, Sex, HIV status,
Treatment received and Name of the referring centers were noted down.

Study period:
From May 2016 to April 2018.

Sampling method: Extra
pulmonary samples (pus, aspirates, body fluids, lymph node [LN] tissue)
were collected in special, plain, universal 30ml clear plastic
container with cap (falcon tubes) under aseptic conditions. 5ml of
samples were received to which buffer solution was added, then the
mixture is loaded to cartridge which were processed by Xpert MTB/RIF
assay (Cepheid-Sunnyvale-USA), as per the guidance document given by
Central TB division, Government of India (RNTCP, 2013; RNTCP, 2012).
The results can be read as MTB detected, MTB not detected, RIF
resistance detected; RIF resistance not detected; RIF resistance
indeterminate; or invalid/error with the help of positive beacons.

Inclusion criteria:
Extra pulmonary samples (pus, aspirates, body fluids, LN tissue) from
all the age group irrespective of gender.

Exclusion criteria:
Blood samples, urine samples, sputum samples and any other EP sample
contaminated with blood is not included in our study.

Results

Total of 281 extrapulmonary samples received during study period,
165(58.7%) were males and 116(41.2%) were females. Most of the cases
were of age group 21 to 30 years. Out of 281 samples received,
73(25.97%) were of FNAC, 115(40.92%) Pleural fluid, 26(9.25%) Ascitic
fluid, 36(12.8%) CSF, 22(7.82%) Pus and 9(3.2%) were aspirates (other
body fluids like synovial fluid, vitreous humor etc) as shown in the
table-1.

Among 281 samples, 67(23.8%) were positive and 214(76.1%) were negative
for MTB as shown in figure-1. Out of 67 positives, 66(98.5%) were
MTB-positive/RIF resistance-negative and 1(1.49%) was MTB- positive/RIF
resistance-positive (in aspirate sample) as shown in the
figure-2. Maximum number of MTB was detected in the age group
of 20-30 years (n=23, 34.3%). Among 165 males and 116 females, MTB was
detected in 44(26.6%) and 23(19.8%) respectively as shown in figure-3.
Out of total 281 patients, 24(8.54%) were HIV positive. Of these 24,
only 8(33.3%) were found positive for MTB in different samples as shown
in the table-2. Among 257 non-HIV patients, MTB was detected in
59(22.9%) as shown in table-3. Among different samples received,
Pleural fluid was in maximum number n=115(40.9%). Maximum MTB positives
were found in FNAC samples n=35(52.2%) as shown in the figure-4.

Table-1: Different EPTB
samples received and their percentages

Specimen type

Frequency

Percentage

FNAC

73

25.97%

Pleural fluid

115

40.92%

Ascitic fluid

26

09.25%

CSF

36

12.81%

Pus

22

07.82%

Aspirates

09

03.20%

Table-2: MTB detected in
different samples received from HIV patients

Samples

Total Positives

Percentage

FNAC

05

62.5%

Pleural fluid

01

12.5%

Ascitic fluid

00

00.0%

CSF

00

00.0%

Pus

02

25.0%

Aspirates

00

00.0%

Table 3: MTB positives
among HIV and Non-HIV patients

HIV status

Total

Mtb positive

Percentage

Positive

24

8

33.3%

Negative

257

59

22.9%

Figure 1:
CBNAAT Results

Figure 2: RIF
Resistance

Figure 3: Age
& sex distribution of patients

Figure 4: CBNAAT
results among different EPTB Patients

Discussion

Extra pulmonary tuberculosis constitutes 20% of burden of TB globally.
EPTB is a pauci-bacillary disease as number of bacteria are less.
Conventional methods (histology and smear microscopy) are not
diagnostic and diagnostic methods (culture methods) are time consuming.
Therefore, a need for new and rapid diagnostic methods, nucleic acid
amplification techniques like GeneXpert (CBNAAT) [7].

Numerous studies have assessed the yield of PCR techniques for the
diagnosis of EPTB [8,9,10].Sanjay G M et al., conducted a study on
trends of EPTB and found higher detection of EPTB cases in younger age
group [11]. Similar results were found in our study. Maximum cases were
in the age group 21-30 years were MTB was detected in 23 (34.3%) cases.

Our several findings are consistent with TB details of other studies
like Balasubramanian et al., where males have higher MTB diagnostic
rates compared to females [12].

A study conducted by Ullah I et al., on EPTB using CBNAAT detected MTB
in 60 (35.7 %) samples, similar to our study where MTB detected in
67(23.8%). CBNAAT showed, 86% of Sensitivity, 88.4% of specificity and
71.7% of positive predictive value while negative predictive value was
high i.e 95.1 % in their study [13].

Scott L E et al., conducted a study on diagnostic accuracy of CBNAAT
for EPTB specimens taking culture as reference, CBNAAT's overall
sensitivity was low (59%) while specificity was high (92%) and Highest
sensitivities was obtained for pus (91%) followed by lymph node
aspirates (80%). Study also found that CBNAAT is not much affected by
bacterial contamination reducing diagnostic delay compared to
traditional methods like culture [14].

Sanjay G M et al found maximum cases of pleural effusion-133(49.81%)
with MTB detected in 13(9.77%). In our study maximum MTB cases were
detected in FNAC samples-35(47.9%) of total 73 cases. Other samples
processed were 115 cases of pleural fluid in which MTB detected in
13(11.3%), 22 pus samples with MTB detected in 8(36.3%), 26 ascitic
fluid in which MTB detected in 2(7.69%), 36 CSF samples with MTB
detection in 4(11.1%) and 9 aspirates where 5(55.5%) were detected
positive for MTB.

In a study done by Armand et al, among individual extrapulmonary
samples, the sensitivity of CBNAAT was highest among lymph nodes
(94.7%). Inclusion of CBNAAT in the initial diagnosis of tubercular
lymphadenopathy in addition to the FNAC would decrease the over
diagnosis of tuberculosis and injudicious use of anti-tuberculosis
treatment [15]. Similarly, in our study maximum MTB was detected in
FNAC of LN samples.

Mittal M et al., on comparison of diagnostic yield of CBNAAT and ZN
(Ziehl-Neelsen) staining from HIV and non-HIV patients with
extra-pulmonary tuberculosis in their study showed of 81 extra
pulmonary samples processed, 21(25.9%) from HIV and 60(74%) from
non-HIV patients. Out of 21 HIV patients, 4(19.05%) and 9(42.85%) were
positive for ZN stain and GeneXpert respectively. Among 60 non-HIV
patients 7(11.66%) and 19 (31.66%) were positive for ZN stain and
GeneXpert respectively. Studies found that CBNAAT is more sensitive
than ZN staining in EPTB while both HIV and non-HIV patients have a
same yield [16]. In our study MTB was detected in 8 out of 24(33.3%)
HIV patients while in non-HIV patients, MTB detected in 59 out of
257(22.9%).

Molecular techniques have subsequently changed the field of
tuberculosis diagnosis in both pulmonary and extra-pulmonary cases and
have been proven to yield rapid results [17].

Conclusion

CBNAAT is a rapid test to confirm presence of MTB with simultaneous
detection of rifampicin resistance in EPTB. Before the advent of
CBNAAT, diagnostic test for all clinically suspected EPTB was staining
method. Its specificity was very low because of which all clinically
suspected EPTB cases use to receive empirical ATT which was a burden on
both patient and health care system. Introduction of CBNAAT for EPTB,
has an impact on early detection, treatment and outcome as most
presumptive cases have a confirmed diagnosis. It has dramatically
reduced the treatment abuse in suspected cases because of lack of
confirmatory tests. This study also helped to know epidemiology of our
locality.